Bacterial antibiotic efflux transporters are important players in conferring intrinsic and acquired resistance to antimicrobials. According to the genomic sequence, Campylobacter jejuni, a leading bacterial cause of foodborne diarrhea in the United States and an agent included in the NIAID Category B Priority Pathogens list, harbors multiple antibiotic efflux transporters of different families. During the previous application period, we determined the function and regulation of two efflux pumps (CmeABC and CmeDEF) of the resistance- nodulation-division (RND) family in Campylobacter. Our findings indicate the efflux system not only contributes to antimicrobial resistance, but also has important physiological functions in facilitating Campylobacter colonization in animal intestinal tract. We have also found that CmeR, a transcriptional factor, represses cmeABC and that bile salts (normally present in the gut) induce the expression of cmeABC by inhibiting the binding of CmeR to the promoter of cmeABC. Our recent preliminary studies also strongly suggest that CmeR is a global regulator and modulates the expression of the MF (major facilitator) and MATE (multidrug and toxic compound extrusion) transporters as well as the C4-dicarboxylate transporters potentially involved in Campylobacter adaptation to oxygen-limited environments, which exist in the niches occupied by Campylobacter in animal hosts. These findings clearly indicate that the modulated expression of the antibiotic efflux system plays important roles in antimicrobial resistance and in facilitating Campylobacter adaptation to environmental changes. Despite these recent advances, the majority of the CmeR-regulated efflux transporters in Campylobacter have not been functionally characterized, and the molecular mechanisms governing the expression of the transporters and the structural basis of CmeR regulation remain to be determined. To close these important gaps in our understanding of the active efflux system in Campylobacter, we plan to pursue 3 specific aims in this application to 1) determine the regulatory mechanisms and functions of the MF and MATE transporters in C. jejuni, 2) define the roles of the CmeR-regulated C4-dicarboxylate transport system in facilitating Campylobacter adaptation to oxygen-limited environments, and 3) elucidate the structural basis of CmeR regulation and the induction mechanisms of bile salts using X-ray crystallography. The proposed studies take advantage of unique resources available in our laboratory and utilize contemporary molecular, genetic, and biochemical approaches as well as an established animal model. Once completed, the proposed work together with the studies conducted in the previous application period will reveal novel information on the functions and regulatory mechanisms of antibiotic efflux transporters in bacteria. The findings will also help to identify potential molecular targets for the control and treatment of antibiotic resistant Campylobacter.